ferrostatin-1 alleviates lipopolysaccharide-induced acute lung … · 2020. 2. 27. · research...

RESEARCH Open Access

Ferrostatin-1 alleviates lipopolysaccharide-induced acute lung injury via inhibitingferroptosisPengfei Liu1,2* , Yetong Feng3, Hanwei Li1,4, Xin Chen5, Guangsuo Wang6, Shiyuan Xu4, Yalan Li7,2* andLei Zhao1,2*

* Correspondence: [email protected]; [email protected]; [email protected] of Anesthesiology, The2nd Clinical Medical College(Shenzhen People’s Hospital) ofJinan University, The 1st AffiliatedHospitals of Southern University ofScience and Technology, Shenzhen518020, China7Department of Anesthesiology,First Affiliated Hospital of JinanUniversity, Guangzhou 510632,ChinaFull list of author information isavailable at the end of the article

Abstract

Background: Ferroptosis is a newly recognized type of cell death, which is differentfrom traditional necrosis, apoptosis or autophagic cell death. However, the positionof ferroptosis in lipopolysaccharide (LPS)-induced acute lung injury (ALI) has notbeen explored intensively so far. In this study, we mainly analyzed the relationshipbetween ferroptosis and LPS-induced ALI.

Methods: In this study, a human bronchial epithelial cell line, BEAS-2B, was treatedwith LPS and ferrostatin-1 (Fer-1, ferroptosis inhibitor). The cell viability was measuredusing CCK-8. Additionally, the levels of malondialdehyde (MDA), 4-hydroxynonenal(4-HNE), and iron, as well as the protein level of SLC7A11 and GPX4, were measuredin different groups. To further confirm the in vitro results, an ALI model was inducedby LPS in mice, and the therapeutic action of Fer-1 and ferroptosis level in lungtissues were evaluated.

Results: The cell viability of BEAS-2B was down-regulated by LPS treatment, togetherwith the ferroptosis markers SLC7A11 and GPX4, while the levels of MDA, 4-HNE andtotal iron were increased by LPS treatment in a dose-dependent manner, whichcould be rescued by Fer-1. The results of the in vivo experiment also indicated thatFer-1 exerted therapeutic action against LPS-induced ALI, and down-regulated theferroptosis level in lung tissues.

Conclusions: Our study indicated that ferroptosis has an important role in theprogression of LPS-induced ALI, and ferroptosis may become a novel target in thetreatment of ALI patients.

Keywords: Ferrostatin-1, Ferroptosis, Lipopolysaccharide, Acute lung injury

BackgroundAcute lung injury (ALI) is regarded as a kind of critical clinical syndrome. It is

also a disorder of acute inflammation, which causes interstitial edema, the accu-

mulation of neutrophils as well as injury of the alveolar epithelium in the lung tis-

sues [1–3]. Numerous studies have indicated that gram-negative bacterial

infections are among the most important causes of ALI, and lipopolysaccharide

(LPS) can lead to the lung injury and inflammatory response, which acts as the

major component of outer membranes of gram-negative bacteria [4–7]. In recent

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, andindicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Cellular & MolecularBiology Letters

Liu et al. Cellular & Molecular Biology Letters (2020) 25:10 https://doi.org/10.1186/s11658-020-00205-0

http://crossmark.crossref.org/dialog/?doi=10.1186/s11658-020-00205-0&domain=pdf

http://orcid.org/0000-0003-0172-189X

mailto:[email protected]





http://creativecommons.org/licenses/by/4.0/

http://creativecommons.org/publicdomain/zero/1.0/

http://creativecommons.org/publicdomain/zero/1.0/

years, LPS has been most widely used in the field of drug-associated ALI models,

which can effectively induce a neutrophilic inflammatory response with an increase

in intrapulmonary cytokines. In addition, LPS is considered as a potent activator of

the innate immune responses via TLR4 pathways. Thus, the use of LPS provides

information about the effects of host inflammatory responses, which occur in bac-

terial infections [8, 9]. Researchers have demonstrated that the intratracheal admin-

istration of LPS can induce the production of inflammatory mediators and reactive

oxygen species (ROS), and worsen the lung tissue injury in an experimental animal

model of ALI [10–13]. Therefore, the development of a novel treatment mode

against LPS-induced ALI, which is based on inhibition of inflammation and oxida-

tive stress, has attracted scientists’ attention in both clinical and pre-clinical

studies.

Different from apoptosis, necrosis or autophagic cell death, ferroptosis is considered

as a novel type of cell death, which mainly results from iron-dependent lipid peroxida-

tion, and is characterized by mitochondrial shrinkage. Emerging evidence suggests that

ferroptosis can be induced by down-regulation of system Xc− activity, inhibition of

glutathione peroxidase 4 (GPX4), and an increase of lipid ROS [14–17]. Many diseases

have been demonstrated to be associated with ferroptosis, such as Alzheimer’s disease

[18], carcinogenesis [19, 20], intracerebral hemorrhage [21], traumatic brain injury [22],

stroke [23], and ischemia-reperfusion injury [24]. In addition, the relationship between

ferroptosis and lung injury or other lung diseases has been investigated by some groups

recently. In 2019, Li et al. found that ferroptosis holds a key role in radiation-induced

lung fibrosis. Their results indicated that liproxstatin-1, a ferroptosis inhibitor, could

alleviate radiation-induced lung fibrosis via down-regulation of TGF-β1 and activation

of the Nrf2 signaling pathway, providing a novel therapeutic target for patients with

radiation-induced lung fibrosis. Moreover, they also investigated the position of ferrop-

tosis in the process of acute radiation-induced lung injury. Their study showed that

obvious ferroptotic characteristic changes of mitochondria were observed in the acute

radiation-induced lung injury model, and the level of glutathione peroxidase 4, a key

marker of ferroptosis, was also decreased in this model, and it could be significantly

alleviated by a ferroptosis inhibitor [25, 26]. Therefore, ferroptosis also played a crucial

role in the acute radiation-induced lung injury. However, the detailed position of

ferroptosis is still unclear for us in LPS-induced ALI.

In the present study, we mainly analyzed the role of ferroptosis in LPS-induced ALI

in vitro and in vivo. We found that ferroptosis could play a critical role in LPS-induced

ALI, and the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively alleviated LPS-induced

ALI. Therefore, our study provided more insights into the cell death pathways in LPS-

induced ALI and established a novel therapeutic approach for patients with ALI.

MethodsCell culture

Cells from the human bronchial epithelial cell line BEAS-2B (ATCC, USA) were

cultured with BEGM Bronchial Epithelial Cell Growth Medium BulletKit (Lonza) in

a humidified incubator at 37 °C with 5% CO2. In addition, the culture medium was

changed every other day. BEAS-2B cells were passaged (dilution, 1:3) every 3 or 4

Liu et al. Cellular & Molecular Biology Letters (2020) 25:10 of 14

days. In addition, air-liquid interface culture of BEAS-2B cells was performed as

the reference [27].

Cell viability assay

To evaluate cell viability, the CCK-8 (Dojindo) method was used in our study as the

references [28, 29]. In brief, BEAS-2B cells were seeded into a 96-well plate at the con-

centration of 5 × 104 cells/well. The cells were cultured for 24 h, then treated with LPS

(Sigma) and Fer-1 (Sigma) in different concentrations for 16 h followed by the addition

of 20 μl of CCK-8 solution directly into the medium (200 μl per well) and incubation at

37 °C for 4 h. The absorbances (Abs) in different groups were detected at 450 nm (n =

3). In the blank group, the well only contained medium, and the cells without any treat-

ment were used as the control group. Herein, the cell viability = (Abs of experimental

group-Abs of blank group)/(Abs of control group-Abs of blank group) × 100%.

Western blot

In our study, the cell samples were lysed using radioimmunoprecipitation assay lysis

buffer (RIPA, Thermo Fisher Scientific), and the total protein concentration of different

groups was detected using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

In our study, the cell lysates (20 μg/lane) were separated using 10% SDS-PAGE gel and

then transferred to nitrocellulose membranes. The membrane was blocked with 5%

nonfat dried milk diluted in PBS, and further incubated with primary antibodies over-

night at 4 °C. Herein, the different primary antibodies used were: anti-SLC7A11 (1:

3000; Cell signaling, Cat #: 12691), anti-GPX4 (1:1000; Santa Crus, Cat #: sc-166,570),

anti-FTH (1:2000; Abcam, Cat #: ab65080) and anti-GAPDH (1:3000; Santa Cruz, Cat

#: sc-47,724). The secondary antibodies used were: Anti-mouse IgG (HRP-conjugated;

1:5000; Sigma-Aldrich, Cat #: A-9044) and anti-rabbit IgG (HRP-conjugated; 1:5000;

Sigma-Aldrich, Cat #: A-0545). Finally, the protein bands in each lane were visualized

using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scien-

tific) and ChemiDoc Imagers (Bio-Rad Laboratories). The results were finally quantified

using the ImageJ 1.x software (National Institutes of Health). All of the raw, uncropped

blots for images throughout the paper are shown in Supplementary Fig. 1.

Evaluation of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and iron level

In our study, to evaluate the ferroptosis level in different groups, the MDA, 4-HNE and

iron levels were detected in each group. The MDA concentration, 4-HNE concentra-

tion and iron concentration in cell lysates were assessed using the Lipid Peroxidation

(MDA) Assay Kit (Sigma-Aldrich, Cat #: MAK085), Lipid Peroxidation (4-HNE) Assay

Kit (Abcam, Cat #: ab238538) and Iron Assay Kit (Sigma-Aldrich, Cat #: MAK025) ac-

cording to the manufacturer’s instructions.

Real-time quantitative PCR (qRT-PCR)

The total RNA was extracted using TRIzol solution (Thermo Fisher Scientific). The

cDNA of different samples was synthesized using 2 μg of total RNA as well as the Tran-

scriptor first-strand cDNA synthesis kit (Promega). Then the qRT-PCR was performed


with SYBR Green Master Mix (TAKARA). The sequences of different primers are as

follows (5′ to 3′):

Mouse Hepcidin -F 5CTGCGCCTTTTCAAGGATGG.

Mouse Hepcidin-R AATTGTTACAGCATTTACAGCAGAAGA.

Mouse Ptgs2-F CTGCGCCTTTTCAAGGATGG.

Mouse Ptgs2-R GGGGATACACCTCTCCACCA.

Mouse Actb-F AAATCGTGCGTGACATCAAAGA.

Mouse Actb-R GCCATCTCCTGCTCGAAGTC.

Human HEPCIDIN-F CTGACCAGTGGCTCTGTTTTC.

Human HEPCIDIN-R GAAGTGGGTGTCTCGCCTC.

Human ACTB-F CCCAGAGCAAGAGAGG.

Human ACTB-R GTCCAGACGCAGGATG.

Animal experiments

In our study, the male C57BL/6 mice were divided randomly into 4 groups (n = 4 per group,

8–10weeks old, weight = 23–25 g): the control group receiving 0.9% NaCl (containing 0.1%

DMSO), the LPS group receiving LPS plus 0.9% NaCl (containing 0.1% DMSO), the Fer-1

group receiving Fer-1 only, and the LPS + Fer-1 group receiving both Fer-1 and LPS. The

LPS-induced ALI model was induced by instilling intratracheally 50 μl of LPS solution (0.2

g/L), then Fer-1 (0.8mg/kg) was administered after LPS challenge via tail vein injection.

The Fer-1 was dissolved in DMSO first, and diluted with 0.9% NaCl. The final concentra-

tion of Fer-1 and DMSO was 0.2mg/ml and 0.1% respectively. After the treatments for 16

h, the mice in each group were euthanized and bronchoalveolar lavage (BAL) fluid was col-

lected via lung lavage. To analyze the differential BAL cell counts, the cells were concen-

trated using a Cytospin 4 (Thermo Fisher Scientific). Cell staining was performed using the

Shandon Kwik-Diff kit (Thermo Fisher Scientific). Additionally, the total protein concentra-

tion and the levels of IL-6 and TNF-α in each sample were detected with the Pierce BCA

Protein Assay Kit (Thermo Fisher Scientific), IL-6 ELISA Kit ELISA kit (Invitrogen) and

TNF-α ELISA Kit (Invitrogen) according to the manufacturer’s instructions. Lung tissues in

different groups were collected for qPCR and western blot detection, and part of lung tis-

sues was fixed using 10% buffered formalin, then the tissues were embedded in paraffin for

histological analyses as the references [25, 30–32]. Herein, a scoring system of 0–4 was used

for the evaluation of lung injury as the reference [33].

Statistical analysis

In this study, all of the results are presented as mean ± SD. SPSS 17.0 software was used

for statistical analysis. Herein, the difference between two groups was analyzed with un-

paired Student’s t-test, and the difference among three or more groups was analyzed

with one-way ANOVA with Bonferroni’s correction. A one-tailed test was used in

Student’s t-test. p < 0.05 was considered statistically significant.

ResultsLPS treatment promotes ferroptosis in BEAS-2B cells

To evaluate the effect of LPS treatment on ferroptosis, BEAS-2B cells were treated with

LPS in different concentrations (1, 5 and 10 mg/L) for 16 h. Cell viability was detected


using the CCK-8 method. The results showed that LPS treatment could inhibit cell

viability in a dose-dependent manner (Fig. 1A). Also, the amount of MDA, 4-HNE and

total iron in the cells treated with LPS was increased significantly (Fig. 1b-d). Some re-

ports have indicated that LPS induces iron overload in vivo and in vitro [34, 35], and

the up-regulation of HEPCIDIN could be the key mechanism during this process. We

detected the level of HEPCIDIN and ferritin heavy chain (FTH) in this study, and the

results indicated that expression of HEPCIDIN was increased in BEAS2B cells treated

with LPS. However, no significant difference in FTH expression was found between the

control group and LPS treatment groups (Fig. 1e-f). Therefore, the iron overload should

be the key reason for up-regulation of total iron. In addition, the protein levels of two

ferroptosis markers, SLC7A11 and GPX4, were also evaluated by western blot. The

results indicated that the expression of both SLC7A11 and GPX4 was down-regulated

by LPS treatment, suggesting that LPS treatment promotes ferroptosis in BEAS-2B cells

(Fig. 1f).

Fer-1 attenuates LPS-induced cell injury via inhibiting ferroptosis

To further confirm the effect of LPS on ferroptosis regulation, Fer-1, a ferroptosis in-

hibitor, was applied in our study. We found that the co-treatment of LPS and Fer-1 still

showed inhibition of cell viability. However, the cell viability in the LPS + Fer-1 group

Fig. 1 The effect of LPS treatment on ferroptosis in BEAS-2B cells. a. Cell viability of BEAS-2B cells treatedwith LPS. The cells were treated with LPS in different concentrations (1, 5, and 10 mg/L) for 16 h, then thecell viability of each group was measured using CCK-8. b-d. Levels of MDA (B), 4-HNE (C) and total iron (D)in the BESA-2B cells treated with LPS. e. mRNA expression of HEPCIDIN. f. Protein levels of SLC7A11 andGPX4 in the BESA-2B cells treated with LPS. Results are expressed as means±SEM (n = 3). *: p < 0.05compared with the 0 mg/L group


was higher than the LPS group, indicating the rescue effect of Fer-1 on LPS-induced

cell death (Fig. 2a). In addition, the amounts of MDA, 4-HNE and total iron in the

LPS + Fer-1 group were also lower than those in the LPS group (Fig. 2b-d). The mRNA

level of HEPCIDIN in the LPS group also could be decreased by Fer-1 treatment

in vitro (Fig. 2e). Moreover, the expression of both SLC7A11 and GPX4 was up-

regulated in the LPS + Fer-1 group compared with the LPS group (Fig. 2f). However,

the treatment with Fer-1 (Fer-1 group) did not affect cell viability or cell ferroptosis in

normal BEAS-2B cells, which could be because of the low basal level of ferroptosis in

normal cells. Overall, those results suggested the key role of ferroptosis in LPS-induced

cell injury.

To simulate the half-way physiological behavior of airway epithelial cells, BEAS-

2B cells grown in an air-liquid interface were used to confirm the role of ferropto-

sis in LPS-induced cell injury. Similar to BEAS-2B cells cultured in normal condi-

tions, the viability of the cells grown in an air-liquid interface was decreased by

LPS treatment, which could be relieved to some degree by Fer-1 (Fig. 3a). More-

over, the levels of MDA, 4-HNE and total iron, as well as the expression of HEP-

CIDIN, in the LPS + Fer-1 group were lower than in the LPS group (Fig. 3b-e), and

the expression of both SLC7A11 and GPX4 was higher in the LPS + Fer-1 group

than the LPS group (Fig. 3f), indicating the rescue effect of Fer-1 in LPS-induced

cell injury.

Fig. 2 Fer-1 attenuates LPS-induced cell injury. a. Cell viability of BEAS-2B cells treated with LPS and Fer-1.The cells were treated with LPS (10 mg/L) and Fer-1 (2 μM) for 16 h, then the cell viability of each groupwas measured using CCK-8. b-d. Levels of MDA (B), 4-HNE (C) and total iron (D) in the BESA-2B cells treatedwith LPS. e. mRNA expression of HEPCIDIN. f. Protein levels of SLC7A11 and GPX4 in the BESA-2B cellstreated with LPS. Results are expressed as means±SEM (n = 3). *: p < 0.05 compared with the control group.#: p < 0.05 compared with the LPS group


Therapeutic action of Fer-1 against LPS-induced ALI

The therapeutic action of Fer-1 against LPS-induced ALI was further evaluated in vivo

using a mouse model. The mice were exposed to an LPS-induced model of inflamma-

tory lung injury, and both BAL fluid and lung tissues were collected for evaluation. The

results indicated that the mice in LPS groups exhibited the greatest degree of injury,

followed by the LPS + Fer-1 group. No obvious injury was found in either the control

group or the Fer-1 group (Fig. 4a-b). The levels of BAL protein and the number of BAL

cells were measured, and the results also indicated the relief of the inflammatory response

in the LPS + Fer-1 group compared with the LPS group (Fig. 5a-b), which was further

confirmed by the differential BAL cell counts (Fig. 5c-d), as well as the levels of BAL pro-

inflammatory cytokines IL-6 and TNF-α (Fig. 5e-f). Therefore, these results indicated that

the ferroptosis inhibitor Fer-1 exerts therapeutic action against LPS-induced ALI.

Fer-1 alleviates LPS-induced ALI via inhibiting ferroptosis

The ferroptosis level in lung tissue was evaluated to analyze the effect of Fer-1. The

qPCR results of mouse Ptgs2 (prostaglandin endoperoxide synthase 2), which is a

marker for the assessment of ferroptosis in vivo, suggested that LPS treatment

promoted ferroptosis in lung tissues, which was alleviated partially by co-treatment

with Fer-1 (Fig. 6a). Similarly, the levels of MDA, 4-HNE and total iron were highest in

Fig. 3 Effect of Fer-1 on LPS-induced cell injury in an air-liquid interface. a. Cell viability of BEAS-2B cells inan air-liquid interface treated with LPS and Fer-1. The cells were treated with LPS (10 mg/L) and Fer-1(2 μM) for 16 h, then the cell viability of each group was measured using CCK-8. b-d. Levels of MDA (B), 4-HNE (C) and total iron (D) in the BESA-2B cells treated with LPS. e. mRNA expression of HEPCIDIN. f. Proteinlevels of SLC7A11 and GPX4 in the BESA-2B cells treated with LPS in an air-liquid interface. Results areexpressed as means±SEM (n = 3). *: p < 0.05 compared with the control group. #: p < 0.05 compared withthe LPS group


the LPS + Fer-1 group, followed by the LPS + Fer-1 group, and Fer-1/control group

(Fig. 6b-d). Similar to the in vitro experiment, the mRNA level of Hepcidin in the LPS

group was also decreased by Fer-1 treatment in vivo (Fig. 6e). Furthermore, the expres-

sion of both SLC7A11 and GPX4 was increased in the LPS + Fer-1 group compared

with the LPS group (Fig. 6f). Collectively, these results indicated that Fer-1 alleviates

LPS-induced ALI via inhibiting ferroptosis, which plays a key role in LPS-induced ALI.

Fig. 4 Therapeutic action of Fer-1 against LPS-induced ALI. a. Hematoxylin and eosin (HE) staining of lungtissue sections from different groups (Scale bar = 50 μm). The raw images of HE staining are shown inSupplementary Fig. 2. b. Lung injury score of mice in each group. Results are expressed as means±SEM(n = 4). *: p < 0.05 compared with the control group. #: p < 0.05 compared with the LPS group

Fig. 5 BAL assay. a. Total protein concentration in BAL fluid from each group. b. Total BAL cell numbers ineach. c-d. Percentage of BAL neutrophils and BAL lymphocytes in different groups. e-f. Concentration of IL-6 and TNF-α in each group. Results are expressed as means±SEM (n = 4). *: p < 0.05 compared with thecontrol group. #: p < 0.05 compared with the LPS group


DiscussionEven though the LPS-induced ALI model has been established for years and widely

used in pre-clinical studies, the accurate mechanisms of LPS-induced ALI are not yet

fully understood [7, 36]. Researchers have found that the excessive accumulation of

ROS and a burst of inflammatory cytokines (e.g. IL-6 and TGF-β) hold an important

position in the pathogenesis of lung injury, and cell death is also considered as a key

issue in LPS-induced ALI. Apoptosis has long been regarded as the major form of cell

death [36–38]. However, because the accumulation of ROS exists in the LPS-induced

ALI, it could be possible that there are still other types of cell death in ALI besides

apoptosis. Ferroptosis is kind of iron-dependent programmed cell death, regulated by

lipid oxidation. This cell death is implicated in many disease pathologies, such as neu-

rodegeneration, inflammation, and ischemia-reperfusion injury [24, 39–41]. In this

study, we mainly explored the position of ferroptosis in LPS-induced ALI. Our results

indicated that the LPS could induce ferroptosis in lung cells in vitro and in vivo, and

the ferroptosis inhibitor showed therapeutic action against LPS-induced ALI, providing

a novel insight into the cell death pathways in LPS-induced ALI.

Moreover, some researchers have demonstrated that all of apoptosis, necroptosis, au-

tophagy, and inflammation were involved in LPS-induced ALI [2, 7, 42, 43]. To further

evaluate each contribution to LPS-induced ALI, the LPS-induced BEAS-2B cell injury

model was established in vitro, and the cells were treated with Fer-1 (2 μM, ferroptosis in-

hibitor), bongkrekic acid (BA, 20 μM, apoptosis inhibitor), necrostatin-1 (Nec-1, 50 μM,

Fig. 6 Fer-1 alleviates LPS-induced ALI through regulating ferroptosis. a. The qPCR analysis of theexpression of Ptgs2 in each group. b-d. Levels of MDA (B), 4-HNE (C) and total iron (D) in the lung tissues ofdifferent groups. e. mRNA expression of HEPCIDIN. f. Protein levels of SLC7A11 and GPX4 in the lung tissuesof different groups. Results are expressed as means±SEM (n = 4). *: p < 0.05 compared with the controlgroup. #: p < 0.05 compared with the LPS group


necroptosis inhibitor), bafilomycin A1 (BAF, 50 nM, autophagy inhibitor), and apocynin

(200 μM, inflammation inhibitor) to rescue cell viability. The results indicated that all of

the inhibitors showed a rescue effect except bafilomycin A1, and apocynin had the best ef-

fect in the LPS-induced injury model in vitro compared with other inhibitors (Fig. 2a and

Fig. 7a-d). Our study mainly indicated that ferroptosis was also involved in LPS-induced

ALI. It could be possible that the therapeutic mechanisms of these inhibitors are related

to each other. For example, the treatment with Fer-1 could decrease the levels of BAL

proinflammatory cytokines IL-6 and TNF-α (Fig. 5e-f). Therefore, it is very hard to evalu-

ate the ratio of contribution from ferroptosis, inflammation, apoptosis and necroptotic cell

death so far. Maybe more specific and effective models are still necessary for the analysis

of each contribution to LPS-induced ALI in vivo.

Fer-1 is the first ferroptosis inhibitor, and is widely used in vitro and in vivo

[44–47]. The function of Fer-1 against ferroptosis mainly depends on the inhibition

of lipid peroxidation. Recently, another group indicated that the anti-ferroptotic ef-

fect of fer-1 is mainly dependent on the scavenging of initiating alkoxyl radicals

and other rearrangement products [48]. We found that the expression level of

HEPCIDIN in the LPS group also could be decreased by Fer-1 treatment in vitro

and in vivo (Fig. 2e, 3e and 6e), which could be a reason for the effect of Fer-1 on

total iron level. However, whether this effect of Fer-1 on hepcidin expression and

Fig. 7 Cell viability of BEAS-2B cells treated with LPS and different inhibitors. Bongkrekic acid (BA, 20 μM,apoptosis inhibitor), necrostatin-1 (Nec-1, 50 μM, necroptosis inhibitor), bafilomycin A1 (BAF, 50 nM,autophagy inhibitor), and apocynin (200 μM, inflammation inhibitor) were used to rescue cell injury inducedby LPS. Results are expressed as means±SEM (n = 3). *: p < 0.05 compared with the control group. #: p <0.05 compared with the LPS group


total iron levels is direct or indirect remains unclear, and the deep mechanisms still

need more investigation in different models. Moreover, some researchers have noted that

the in vivo function of Fer-1 is weaker than the function in vitro, because of the plasma

and metabolic instability [49, 50]. Therefore, the development of a more stable and potent

ferroptosis specific inhibitor is still necessary for the in vitro study in the field of ferropto-

sis. Recently, some researchers found that liproxstatin-1 (another ferroptosis inhibitor) is

more stable than Fer-1, and liproxstatin-1 also did not interfere with other types of cell

death [26, 49, 50]. In our study, Fer-1 was applied in both in vitro and in vivo models, and

showed an obvious effect against ferroptosis. It might be possible that the effect would be

further improved if liproxstatin-1 was used in our research. Also, Fer-1 was administered

after LPS challenge via tail vein injection herein. Therefore, Fer-1 in venous blood will

enter the pulmonary circulation and work on lung tissue after injection immediately,

which will enhance the therapeutic action of Fer-1 compared with intraperitoneal injec-

tion or oral administration. Even though other ferroptosis inhibitors may have a longer

half-life in vivo, no comparative analysis has been performed in detail in a lung injury

model so far. Our results mainly indicated that Fer-1 exerts therapeutic action against

ALI, and it is also possible that the parameters at a shorter time point (less than 16 h after

the injection of Fer-1) could show a better therapeutic effect. Of course, this hypothesis

still needs our further exploration.

Numerous studies have demonstrated the crucial role of the infiltration of inflamma-

tory cells, which is caused by the inflammatory cytokines during the progress of LPS-

induced ALI. Furthermore, some researchers also noted that the increased infiltration

of inflammatory cells could enhance the synthesis and accumulation of ROS in lung tis-

sues [2, 4, 6, 12, 13]. In our study, the levels of IL-6 and TNF-α in BAL were increased

in the LPS-induced ALI, and treatment with the ferroptosis inhibitor Fer-1 decreased

the levels of both IL-6 and TNF-α in BAL, indicating the relationship between ferropto-

sis and inflammatory cytokines. Some studies have indicated that lipid peroxidation in

ferroptosis can promote the inflammation and regulate the level of different inflamma-

tory cytokines [39, 51, 52], which is consistent with our results. Moreover, the excessive

accumulation of ROS also causes oxidative damage and an inflammatory response in

lung tissues [53–55]. Ferroptosis is mainly induced by the failure of membrane lipid re-

pair, and further leads to the increase of ROS on the membrane lipids. Therefore, the

excessive accumulation of ROS caused by LPS treatment could be associated with the

ferroptosis in LPS-induced ALI, and ROS-induced oxidative damage may also be

regarded as a key causative factor in the different inflammatory events involved in ALI.

However, the detailed role of ferroptosis and ROS in the inflammatory micro-

environment still needs be explored intensively.

SLC7A11 and GPX4 are considered as the central regulators of ferroptosis, and

reduced levels of GPX4 and SLC7A11 are always regarded as markers of ferropto-

sis [56–58]. In our study, we found that both SLC7A11 and GPX4 were clearly de-

creased in the LPS-induced ALI model, suggesting that ferroptosis occurred during

the process of LPS-induced ALI. Moreover, the administration of Fer-1 inhibited

LPS-induced ALI and increased the protein levels of both SLC7A11 and GPX4 in

lung cells and tissues. These results further suggested that ferroptosis holds an im-

portant position during LPS-induced ALI, and a ferroptosis inhibitor should have

an effective therapeutic action and reduce the histological alteration in ALI mice.


ConclusionsIn conclusion, our results indicated that ferroptosis played an important role in LPS-

induced ALI, and Fer-1 alleviated LPS-induced ALI and the inflammatory response

in vivo effectively via regulating ferroptosis. Therefore, our study demonstrated that a

novel form of regulated cell death, ferroptosis, occurred in LPS-induced ALI, which

was totally distinct from the classical cell apoptosis; that ferroptosis holds the potential

to become a novel therapeutic target in ALI; and that a ferroptosis inhibitor might be

an effective kind of drug for ALI patients.

Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s11658-020-00205-0.

Additional file 1: Supplementary Fig. 1. Uncropped blots for images shown throughout the paper.

Additional file 2: Supplementary Fig. 2. Raw images of HE staining in Fig. 4a.

Abbreviations4-HNE: 4-hydroxynonenal; Abs: Absorbances; ALI: Acute lung injury; ATCC: American type culture collection;BA: Bongkrekic acid; BAF: Bafilomycin A1; BAL: Bronchoalveolar lavage; Fer-1: Ferrostatin-1; LPS: Lipopolysaccharide;MDA: Malondialdehyde; Nec-1: Necrostatin-1; Ptgs2: Prostaglandin endoperoxide synthase 2; qPCR: Real-timequantitative PCR; RIPA: Radioimmunoprecipitation assay lysis buffer; ROS: Reactive oxygen species

AcknowledgementsNot applicable.

Authors’ contributionsPL, YF, HL and SX conceived and designed the experiments; PL, HL, and YF performed the experiments; YF, XC, GW,HL and PL analyzed the data; GW and LZ contributed reagents/materials/analysis tools; YL, LZ and SX wrote the paper;PL, YL, LZ and SX conceived and supervised the studies. All authors read and approved the final manuscript.

FundingThis study was supported by grants from the National Natural Science Foundation of China (31900547), ShenzhenPeacock Plan (KQTD2016113015442590), Project funded by China Postdoctoral Science Foundation (2019 M653277)and Science and Technology Planning Project of Guangdong Province (2016ZC0244).

Availability of data and materialsAll data generated or analyzed during this study are included in this published article and its supplementaryinformation files.

Ethics approval and consent to participateAll animal experiments were approved by the Committee on the Ethics of Animal Experiments and Human SubjectResearch of Jinan University (Approval number: JU20180923–066) on January 8th 2018. All of the experiments wereperformed in accordance with the Declaration of Helsinki, which the Committee on the Ethics of Animal Experimentsand Human Subject Research of Jinan University acts on.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Anesthesiology, The 2nd Clinical Medical College (Shenzhen People’s Hospital) of Jinan University, The1st Affiliated Hospitals of Southern University of Science and Technology, Shenzhen 518020, China. 2IntegratedChinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, China. 3HealthScience Center, School of Basic Medical Sciences, Shenzhen University, Shenzhen 518037, China. 4Department ofAnesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou 510280, China. 5Department ofLaboratory Medicine, The 2nd Clinical Medicine College (Shenzhen People’s Hospital) of Jinan University, The 1stAffiliated Hospitals of Southern University of Science and Technology, Shenzhen 518020, China. 6Department ofThoracic Surgery, The 2nd Clinical Medicine College (Shenzhen People’s Hospital) of Jinan University, The 1st AffiliatedHospitals of Southern University of Science and Technology, Shenzhen 518020, China. 7Department of Anesthesiology,First Affiliated Hospital of Jinan University, Guangzhou 510632, China.


https://doi.org/10.1186/s11658-020-00205-0

Received: 12 September 2019 Accepted: 17 February 2020

References1. Butt Y, Kurdowska A, Allen TC. Acute lung injury: a clinical and molecular review. Arch Pathol Lab Med. 2016;140(4):345–50.2. Cao C, Yin C, Shou S, Wang J, Yu L, Li X, et al. Ulinastatin protects against LPS-induced acute lung injury by attenuating

TLR4/NF-kappaB pathway activation and reducing inflammatory mediators. Shock. 2018;50(5):595–605.3. He J, Qi D, Tang XM, Deng W, Deng XY, Zhao Y, et al. Rosiglitazone promotes ENaC-mediated alveolar fluid clearance in

acute lung injury through the PPARgamma/SGK1 signaling pathway. Cell Mol Biol Lett. 2019;24:35.4. Hsieh YH, Deng JS, Pan HP, Liao JC, Huang SS, Huang GJ. Sclareol ameliorate lipopolysaccharide-induced acute lung

injury through inhibition of MAPK and induction of HO-1 signaling. Int Immunopharmacol. 2017;44:16–25.5. Zhou Y, Liu T, Duan JX, Li P, Sun GY, Liu YP, et al. Soluble epoxide hydrolase inhibitor attenuates lipopolysaccharide-

induced acute lung injury and improves survival in mice. Shock. 2017;47(5):638–45.6. Yang JX, Li M, Chen XO, Lian QQ, Wang Q, Gao F, et al. Lipoxin A4 ameliorates lipopolysaccharide-induced lung injury

through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial-mesenchymaltransition. Respir Res. 2019;20(1):192.

7. Kolomaznik M, Nova Z, Calkovska A. Pulmonary surfactant and bacterial lipopolysaccharide: the interaction and itsfunctional consequences. Physiol Res. 2017;66(Suppl 2):S147–S57.

8. Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2008;295(3):L379–99.

9. Liang Y, Yang N, Pan G, Jin B, Wang S, Ji W. Elevated IL-33 promotes expression of MMP2 and MMP9 via activatingSTAT3 in alveolar macrophages during LPS-induced acute lung injury. Cell Mol Biol Lett. 2018;23:52.

10. Lv H, Liu Q, Wen Z, Feng H, Deng X, Ci X. Xanthohumol ameliorates lipopolysaccharide (LPS)-induced acute lung injuryvia induction of AMPK/GSK3beta-Nrf2 signal axis. Redox Biol. 2017;12:311–24.

11. Wu G, Dai X, Li X, Jiang H. Antioxidant and anti-inflammatory effects of Rhamnazin on lipopolysaccharide-induced acutelung injury and inflammation in rats. Afr J Tradit Complement Altern Med. 2017;14(4):201–12.

12. Park J, Chen Y, Zheng M, Ryu J, Cho GJ, Surh YJ, et al. Pterostilbene 4′-beta-Glucoside attenuates LPS-induced acutelung injury via induction of Heme Oxygenase-1. Oxidative Med Cell Longev. 2018;2018:2747018.

13. Dang X, Du G, Hu W, Ma L, Wang P, Li Y. Peroxisome proliferator-activated receptor gamma coactivator-1alpha/HSF1axis effectively alleviates lipopolysaccharide-induced acute lung injury via suppressing oxidative stress and inflammatoryresponse. J Cell Biochem. 2019;120(1):544–51.

14. Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, et al. Ferroptosis: a regulated cell death Nexuslinking metabolism, redox biology, and disease. Cell. 2017;171(2):273–85.

15. Xie Y, Hou W, Song X, Yu Y, Huang J, Sun X, et al. Ferroptosis: process and function. Cell Death Differ. 2016;23(3):369–79.16. Cao JY, Dixon SJ. Mechanisms of ferroptosis. Cell Mol Life Sci. 2016;73(11–12):2195–209.17. Lei P, Bai T, Sun Y. Mechanisms of Ferroptosis and relations with regulated cell death: a review. Front Physiol. 2019;10:139.18. Masaldan S, Belaidi AA, Ayton S, Bush AI. Cellular senescence and Iron Dyshomeostasis in Alzheimer's disease.

Pharmaceuticals (Basel). 2019;12(2):E93.19. Toyokuni S. The origin and future of oxidative stress pathology: from the recognition of carcinogenesis as an iron

addiction with ferroptosis-resistance to non-thermal plasma therapy. Pathol Int. 2016;66(5):245–59.20. Wang M, Mao C, Ouyang L, Liu Y, Lai W, Liu N, et al. Long noncoding RNA LINC00336 inhibits ferroptosis in lung cancer

by functioning as a competing endogenous RNA. Cell Death Differ. 2019;26(11):2329–43.21. Chen B, Chen Z, Liu M, Gao X, Cheng Y, Wei Y, et al. Inhibition of neuronal ferroptosis in the acute phase of

intracerebral hemorrhage shows long-term cerebroprotective effects. Brain Res Bull. 2019;153:122–32.22. Kenny EM, Fidan E, Yang Q, Anthonymuthu TS, New LA, Meyer EA, et al. Ferroptosis contributes to neuronal death and

functional outcome after traumatic brain injury. Crit Care Med. 2019;47(3):410–8.23. Zille M, Karuppagounder SS, Chen Y, Gough PJ, Bertin J, Finger J, et al. Neuronal death after hemorrhagic stroke in vitro

and in vivo shares features of Ferroptosis and Necroptosis. Stroke. 2017;48(4):1033–43.24. Guan X, Li X, Yang X, Yan J, Shi P, Ba L, et al. The neuroprotective effects of carvacrol on ischemia/reperfusion-induced

hippocampal neuronal impairment by ferroptosis mitigation. Life Sci. 2019;235:116795.25. Li X, Zhuang X, Qiao T. Role of ferroptosis in the process of acute radiation-induced lung injury in mice. Biochem

Biophys Res Commun. 2019;519(2):240–5.26. Li X, Duan L, Yuan S, Zhuang X, Qiao T, He J. Ferroptosis inhibitor alleviates Radiation-induced lung fibrosis (RILF) via

down-regulation of TGF-beta1. J Inflamm (Lond). 2019;16:11.27. Prodanovic D, Keenan CR, Langenbach S, Li M, Chen Q, Lew MJ, et al. Cortisol limits selected actions of synthetic

glucocorticoids in the airway epithelium. FASEB J. 2018;32(3):1692–704.28. Zhao L, Feng Y, Chen X, Yuan J, Liu X, Chen Y, et al. Effects of IGF-1 on neural differentiation of human umbilical cord

derived mesenchymal stem cells. Life Sci. 2016;151:93–101.29. Liu P, Feng Y, Dong D, Liu X, Chen Y, Wang Y, et al. Enhanced renoprotective effect of IGF-1 modified human umbilical

cord-derived mesenchymal stem cells on gentamicin-induced acute kidney injury. Sci Rep. 2016;6:20287.30. Liu P. Rojo de la Vega M, Sammani S, Mascarenhas JB, Kerins M, Dodson M, et al. RPA1 binding to NRF2

switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression. Proc Natl Acad Sci U S A.2018;115(44):E10352–61.

31. Liu P, de la Vega MR, Dodson M, Yue F, Shi B, Fang D, et al. Spermidine confers liver protection by enhancing NRF2signaling through a MAP 1S-mediated noncanonical mechanism. Hepatology. 2019;70(1):372–88.

32. Zhang B, Wang B, Cao S, Wang Y, Wu D. Silybin attenuates LPS-induced lung injury in mice by inhibiting NF-kappaBsignaling and NLRP3 activation. Int J Mol Med. 2017;39(5):1111–8.

33. Wei F, Wen S, Wu H, Ma L, Huang Y, Yang L. Partial liquid ventilation-induced mild hypothermia improves the lungfunction and alleviates the inflammatory response during acute respiratory distress syndrome in canines. BiomedPharmacother. 2019;118:109344.


34. Le BV, Khorsi-Cauet H, Bach V, Gay-Queheillard J. Modulation of Pseudomonas aeruginosa lipopolysaccharide-inducedlung inflammation by chronic iron overload in rat. FEMS Immunol Med Microbiol. 2012;64(2):255–64.

35. You LH, Yan CZ, Zheng BJ, Ci YZ, Chang SY, Yu P, et al. Astrocyte hepcidin is a key factor in LPS-induced neuronalapoptosis. Cell Death Dis. 2017;8(3):e2676.

36. Jiang K, Zhang T, Yin N, Ma X, Zhao G, Wu H, et al. Geraniol alleviates LPS-induced acute lung injury in mice viainhibiting inflammation and apoptosis. Oncotarget. 2017;8(41):71038–53.

37. Xie W, Lu Q, Wang K, Lu J, Gu X, Zhu D, et al. miR-34b-5p inhibition attenuates lung inflammation and apoptosis in anLPS-induced acute lung injury mouse model by targeting progranulin. J Cell Physiol. 2018;233(9):6615–31.

38. Badshah H, Ali T, Kim MO. Osmotin attenuates LPS-induced neuroinflammation and memory impairments via the TLR4/NFkappaB signaling pathway. Sci Rep. 2016;6:24493.

39. Zhang YH, Wang DW, Xu SF, Zhang S, Fan YG, Yang YY, et al. Alpha-Lipoic acid improves abnormal behavior bymitigation of oxidative stress, inflammation, ferroptosis, and tauopathy in P301S tau transgenic mice. Redox Biol. 2018;14:535–48.

40. Pena-Bautista C, Vento M, Baquero M, Chafer-Pericas C. Lipid peroxidation in neurodegeneration. Clin Chim Acta. 2019;497:178–88.

41. Citron BA, Ameenuddin S, Uchida K, Suo WZ, SantaCruz K, Festoff BW. Membrane lipid peroxidation inneurodegeneration: role of thrombin and proteinase-activated receptor-1. Brain Res. 1643;2016:10–7.

42. Liu H, Yu X, Yu S, Kou J. Molecular mechanisms in lipopolysaccharide-induced pulmonary endothelial barrierdysfunction. Int Immunopharmacol. 2015;29(2):937–46.

43. Li C, Pan J, Ye L, Xu H, Wang B, Xu H, et al. Autophagy regulates the therapeutic potential of adipose-derived stem cellsin LPS-induced pulmonary microvascular barrier damage. Cell Death Dis. 2019;10(11):804.

44. Degterev A, Linkermann A. Generation of small molecules to interfere with regulated necrosis. Cell Mol Life Sci. 2016;73(11–12):2251–67.

45. Doll S, Conrad M. Iron and ferroptosis: a still ill-defined liaison. IUBMB Life. 2017;69(6):423–34.46. Abdalkader M, Lampinen R, Kanninen KM, Malm TM, Liddell JR. Targeting Nrf2 to suppress Ferroptosis and

mitochondrial dysfunction in Neurodegeneration. Front Neurosci. 2018;12:466.47. Bogacz M, Krauth-Siegel RL. Tryparedoxin peroxidase-deficiency commits trypanosomes to ferroptosis-type cell death.

Elife. 2018;7:e37503.48. Miotto G, Rossetto M, Di Paolo ML, Orian L, Venerando R, Roveri A, et al. Insight into the mechanism of ferroptosis

inhibition by ferrostatin-1. Redox Biol. 2019;28:101328.49. Zilka O, Shah R, Li B, Friedmann Angeli JP, Griesser M, Conrad M, et al. On the mechanism of Cytoprotection by Ferrostatin-1

and Liproxstatin-1 and the role of lipid peroxidation in Ferroptotic cell death. ACS Cent Sci. 2017;3(3):232–43.50. Friedmann Angeli JP, Schneider M, Proneth B, Tyurina YY, Tyurin VA, Hammond VJ, et al. Inactivation of the ferroptosis

regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol. 2014;16(12):1180–91.51. Masaldan S, Clatworthy SAS, Gamell C, Meggyesy PM, Rigopoulos AT, Haupt S, et al. Iron accumulation in senescent

cells is coupled with impaired ferritinophagy and inhibition of ferroptosis. Redox Biol. 2018;14:100–15.52. Zhang Q, Wu H, Zou M, Li L, Li Q, Sun C, et al. Folic acid improves abnormal behavior via mitigation of oxidative stress,

inflammation, and ferroptosis in the BTBR T+ tf/J mouse model of autism. J Nutr Biochem. 2019;71:98–109.53. El-Kenawi A, Ruffell B. Inflammation, ROS, and mutagenesis. Cancer Cell. 2017;32(6):727–9.54. Barrows IR, Ramezani A, Raj DS. Inflammation, immunity, and oxidative stress in hypertension-Partners in Crime? Adv

Chronic Kidney Dis. 2019;26(2):122–30.55. Agita A, Alsagaff MT. Inflammation, immunity, and hypertension. Acta Med Indones. 2017;49(2):158–65.56. Imai H, Matsuoka M, Kumagai T, Sakamoto T, Koumura T. Lipid peroxidation-dependent cell death regulated by GPx4

and Ferroptosis. Curr Top Microbiol Immunol. 2017;403:143–70.57. Chen D, Fan Z, Rauh M, Buchfelder M, Eyupoglu IY, Savaskan N. ATF4 promotes angiogenesis and neuronal cell death

and confers ferroptosis in a xCT-dependent manner. Oncogene. 2017;36(40):5593–608.58. Wang Y, Yang L, Zhang X, Cui W, Liu Y, Sun QR, et al. Epigenetic regulation of ferroptosis by H2B monoubiquitination

and p53. EMBO Rep. 2019;20(7):e47563.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


ferrostatin-1 alleviates lipopolysaccharide-induced acute lung … · 2020. 2. 27. · research...

Documents